Nitric oxide synthase (NOS) catalyzes the oxygenation of arginine to nitric oxide (NO) and citrulline. The enzyme is unique in requiring 5 factors for activity, including tetrahydrobiopterin (BH4). One of the many roles proposed for NO is that it acts as a neurotransmitter in the brain, and appears to be involved in the physiology of learning and memory. Clarification of the action of BH4 in nitric oxide formation is therefore important in understanding neurotransmission and some of its abnormal aspects. It is well established that BH4 has marked physical effects on NOS, including the promotion of arginine binding and stabilization of the active dimeric state, etc. However, it is not clear whether or not BH4 acts as a stoichiometric redox reactant in NO synthesis, comparable to its classical role in the aromatic amino acid hydroxylases. As a first step in examining this question, we have demonstrated that during normal multiple turnovers of NOS, NOS-bound BH4 does not remain in a static state, but cycles between the reduced and oxidized forms. BH4 oxidation was increased in the presence of the substrate, arginine. These combined observations suggest that BH4 may be acting as a redox reactant in the oxygenation of arginine to form NO. We have followed up on this proposal by examining whether NOS-bound BH4 is converted to the carbinolamine (4-alpha-hydroxy tetrahydrobiopterin) during NO formation. The carbinolamines (6- and 7-isomers) are known to be formed only as products of BH4 acting as a stoichiometric redox reactant, as exemplified by the aromatic amino acid hydroxylases. Any such formation of the carbinolamine by NOS would therefore provide definitive evidence that a similar stoichiometric role of BH4 also operates for this enzyme. By using specific and sensitive methods designed for this project, we now have preliminary evidence that BH4 bound to NOS is converted to the carbinolamine during NO formation. The factors required for carbinolamine formation were the same as those for NO formation. Furthermore no carbinolamine was detected in the presence of carbinolamine dehydratase, which converts the carbinolamine to quinonoid dihydrobiopterin. We are currently confirming these observations with the goal of completing this project in the very near future.